The present invention relates to a composition useful for early and profuse sporulation in fungi with about 100 fold increase in spore count within 48 hours of inoculation, said composition comprising molasses ranging between 5-20 g/L, Corn Steep Liquor (CSL) ranging between 10-25 g/L, Sodium chloride (NaCl) ranging between 5-15 g/L, calcium sulphate (CaSO4) ranging between 0.1-0.5 g/L, Potassium dihydrogen phosphate (KH2PO4) ranging between 0.001-0.01 g/L, Magnesium sulphate (MgSO4.7H2O) ranging between 0.001-0.01 g/L, Copper sulphate (CuSO4) ranging between 0.001-0.0050 g/L, Ferrous sulphate (FeSO4) ranging between 0.0009-0.005 g/L, an anti-foam agent, and optionally solidifying agent and a method thereof.
Trichoderma species are used as efficient biocontrol agents against the soil-borne Wilt and Root-rot diseases of various crop plants. With the growing awareness of the harmful effects of many synthetic chemical pesticides on humans, non-target animals and plant species, and the environment as a whole, there has been a shift in attention to the research and development of more environment-friendly methods of pest and disease control.
Use of environmentally safe Disease Management Technologies based on Biopesticide and Biocontrol agents through natural enemiesxe2x80x94parasitoids, predators and pathogens in integrated pest management programmes are ever increasing. Biocontrol agents like Trichoderma species and Gliocladium species are examples of some of the biocontrol agents used in the management of root diseases of chickpea, peas, groundnut, soybean spices and vegetables.
Root-rot diseases of crops in general and pulses in particular are a serious problem in rain-fed areas caused mostly by species of Fusarium and Rhizoctonia etc. Chemical control of these diseases requires a continuous use of fungicides, which is not economical and causes deleterious effects on the rhizosphere microflora and also have the residual problems.
WHO now has banned even some of the commonly used fungicides due to their severe toxicity. Under these circumstances the biocontrol agents can be used for the management of Root-rot diseases at comparatively cheaper cost with long term antagonistic effects against the pathogens because these biocontrol agents multiply and remain near the root zone of the plant giving protection to these plants throughout the growth period.
Investigations carried out world over have confirmed, that synthetic plant protection chemicals such as fungicides, insecticides and herbicides deteriorate soil fertility. Such deterioration occurs through disappearance of bio-diversity in soils. On account of this, several strains of soil microorganisms have been used which are able to suppress a wide range of disease causing microorganisms.
Jackson et al., (Enzyme-Microb. Technol.; (1991) 13, 6, 456-61) reported the culture media optimisation for production of the biological control agents Gliocladium virens G20, Trichoderma pseudokoningii IMI 322662, and T. viride IMI 322659 and IMI 322663. In glucose-alanine medium, the optimum dry wt./g carbon occurred with a C:N ratio of 15:1. Addition of 3.28-mg atoms iron/l to the medium increased biomass production of all isolates, but a concentration of 164-mg atoms/l was toxic to the Trichoderma species. Growth decreased in media lacking Mg, P, K or S, but the amount of the decrease differed between the four isolates. Sporulation in agar was reduced in the absence of Mg, P, K and N. Addition of biotin, p-amino -benzoic acid and thiamine-HCl increased biomass production slightly. The glucose-alanine basal medium supported better growth of all 4 isolates than a commercial molasses-yeast medium; conidia production was greater in the molasses-yeast medium. The pH of the glucose-alanine medium remained constant at 4.5, whereas the pH of the yeast-molasses medium (initially 5.5) increased to 8.0-8.6. Chlamydospores were produced by all isolates, but the numbers varied according to the culture conditions used.
Gervais and Sarrette (J.Fement.Bioeng. (1990, 69, 1, 46-50) reported the Emerson agar medium (18 g/l agar) containing sodium octanoate (1 g/l) as a 2-heptanone precursor and glycerol as a water activity depressor for the solid-state fermentation of T. viride for cheese aroma production. Cultures were grown in Petri dishes at 20xc2x0 C. Sporulation was visible on the 9th day.
Toyama et al (J. Ferment. Technol.; (1983, 61, 4, 409-11) reported a sporulation medium for T. reesei QM 9414, to be used for the production of protoplasts. Kennedy-M-J; Davies-R-J; Surrey-M-R; Reader-S-L; Hoefakker-P-C, Australia""s. Biotechnol; (1995) 5, 6, 349-54 reported preparation of biological control agents based on Serratia entomophila, Bacillus thuringiensis, Pestalotia species, Truncatella augustata, Trichoderma viride, Trichoderma species., cricket-paralysis virus, flock-house virus. The report focuses on the 4 areas of process expertise required to develop and produce a commercial biological control agent: (a) culture medium design, (b) scale-up of the biological production system, (c) downstream processing of the product and (d) extending the shelf life of the biological control agent.
Nigam P. (Process-Biochem.; (1994, 29, 5, 337-42) reported a medium containing diluted molasses solutions of 3-4% sugar concentration for T. viride QM 9414, T. reesei Rut-C-30 NRRL 11460 under submerged fermentation conditions (SF) performed in flasks in 100 ml medium with agitation at 180 rpm for 5 days.
Culture media for two-stage culture of T. longibrachiatum, T. viride, T. aureoviride, AN:
91-13819, CA: Moscow-Tech.Inst.Food-Ind. were reported. The culture medium (pH 4.0-4.2) used in stage I had the following composition (wt. %): wheat bran, 0.3-0.4; additional hydrolysed cotton cake, 0.3-0.4; gibbersib (sic) biomass, 0.45-0.50; NH4H2PO4, 0.25-0.30; K2SO4, 0.20-0.22; and MgSO4, 0.025-0.030. The culture medium used in stage 11 (pH 5.0-5.2) had the following composition (wt. %): beet-root pulp, 2.5-2.8; wheat bran, 0.3-0.4; additional hydrolysed cotton cake, 0.3-0.4; K2SO4, 0.25-0.30; NH4NO3, 0.25-0.30 and stomach contents hydrolyzate containing reducing compounds, 0.20-0.25.
Abou-Zeid (Bioresource-Technol.; (1991) 37, 3, 239-42) reported the following medium (pH 6) (g/l): cellulosic-source, 10.0; (NH4)2SO4, 2.0; KH2PO4, 1.0; KCl, 0.5; MgSO4.7H2O, 0.5; MnSO4.4H2O, 0.05; and FeSO4.7H2O, 0.005, for the Fermentation of T. viride using leaflets and midribs of date palm (Phoenix dactylifera) leaves dried at 100-105xc2x0 C., 144 hr at 30xc2x0 C. with 200 rpm agitation. Pre-treatment of palm leaflets with 1% NaOH and 5% H2SO4 improved their suitability as C-sources for T. viride growth.
The conventional media reported earlier are based on expensive gradients such as yeast extract, malt extract, protein hydrolysates and the like. Such a media are not suited for the economic mass production of these biocontrol agents by fermentation at technical scales.
We have designed medium for the mass production of spores of Trichoderma species, a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and other scantily sporulating species of fungi under solid state, surface and submerged fermentation conditions based on inexpensive agro-based industrial by-products like beet-root pulp, wheat bran, corn-steep liquor and molasses. These agro-waste industrial by products are rich in micro-nutrients and other essential elements like sugars, amino acids providing synergistic effects on the growth and sporulation of these fungi.
The main object of the present invention is to develop a composition for early sporulation of the fungi.
Another main object of the present invention is to develop a composition for profuse sporulation of the fungi.
Yet another object of the present invention is to develop a composition for early and profuse sporulation in Trichoderma species, Aspergillus species, and Penicillium species.
Still another object of the present invention is to develop a composition for early and profuse sporulation in scantily sporulating fungi.
Still another object of the present invention is to develop a composition much more economical than commercially available compositions.
Still another object of the present invention is to develop a method for early and profuse sporulation in fungi.
Still another object of the present invention is to develop a method for sporulation under solid state, surface, or submerged fermentation conditions.
The present invention relates to a composition useful for early and profuse sporulation in fungi with about 100 fold increase in spore count within 48 hours of inoculation, said composition comprising molasses ranging between 5-20 g/L, Corn Steep Liquor (CSL) ranging between 10-25 g/L, Sodium chloride (NaCl) ranging between 5-15 g/L, calcium sulphate (CaSO4) ranging between 0.1-0.5 g/L, Potassium dihydrogen phosphate (KH2PO4) ranging between 0.001-0.01 g/L, Magnesium sulphate (MgSO4.7H2O) ranging between 0.001-0.01 g/L, Copper sulphate (CuSO4) ranging between 0.001-0.0050 g/L, Ferrous sulphate (FeSO4) ranging between 0.0009-0.005 g/L, an anti-foam agent, and optionally solidifying agent and a method thereof.
Accordingly, the present invention relates to a composition useful for early and profuse sporulation in fungi, said composition comprising molasses ranging between 5-20 g/L, Corn Steep Liquor (CSL) ranging between 10-25 g/L, Sodium chloride (NaCl) ranging between 5-15 g/L, calcium sulphate (CaSO4) ranging between 0.1-0.5 g/L, Potassium dihydrogen phosphate (KH2PO4) ranging between 0.001-0.01 g/L, Magnesium sulphate (MgSO4.7H2O) ranging between 0.001-0.01 g/L, Copper sulphate (CuSO4) ranging between 0.001-0.0050 g/L, Ferrous sulphate (FeSO4) ranging between 0.0009-0.005 g/L, an anti-foam agent, and optionally solidifying agent.
In an embodiment of the present invention, said composition show most efficient sporulation with composition comprising molasses about 7.50 g/L, Corn Steep Liquor (CSL) about 20 g/L, Sodium chloride (NaCl) about 10 g/L, calcium sulphate (CaSO4) about 0.25 g/L, Potassium dihydrogen phosphate (KH2PO4) about 0.0060 g/L, Magnesium sulphate (MgSO4.7 H2O) about 0.0050 g/L, Copper sulphate (CuSO4) about 0.0010 g/L, Ferrous sulphate (FeSO4) about 0.0016 g/L, an anti-foam agent, and optionally solidifying agent.
In yet another embodiment of the present invention, using silicon oil as an anti-foam agent.
In still another embodiment of the present invention, silicon oil ranging between 0.1-0.5 ml.
In still another embodiment of the present invention, using agar as a solidifying agent.
In still another embodiment of the present invention, agar ranging between 5-25 g/L.
In still another embodiment of the present invention, said composition is effective with fungi selected from a group comprising Trichoderma species, Aspergillus species, and Penicillium species.
In still another embodiment of the present invention, said composition is effective with fungi Trichoderma species.
In still another embodiment of the present invention, said composition shows about 100-fold increase in spore count within 48 hours of inoculation.
In still another embodiment of the present invention, said composition shows average spore count ranging between 1-5xc3x97108 conidiospores/ml.
In still another embodiment of the present invention, said composition shows percentage viability of generated spores ranging between 90 to 100%.
In still another embodiment of the present invention, said composition is equally effective with scantily sporulating fungi.
In still another embodiment of the present invention, said composition is used for mass production of fungi of biocontrol activity.
In still another embodiment of the present invention, said composition is economical as compared to other commercially available compositions for same purpose.
In a further embodiment of the present invention a method for early and profuse sporulation in fungi with about 100 fold increase in spore count within 48 hours of inoculation, said method comprising:
In another embodiment of the present invention, inoculating about one week old spore culture of the fungi in a medium comprising molasses ranging between 5-20 g/L, Corn Steep Liquor (CSL) ranging between 10-25 g/L, Sodium chloride (NaCl) ranging between 5-15 g/L, calcium sulphate (CaSO4) ranging between 0.1-0.5 g/L, Potassium dihydrogen phosphate (KH2PO4) ranging between 0.001-0.01 g/L, Magnesium sulphate (MgSO4.7H2O) ranging between 0.001-0.01 g/L, Copper sulphate (CuSO4) ranging between 0.001-0.0050 g/L, Ferrous sulphate (FeSO4) ranging between 0.0009-0.005 g/L, an anti-foam agent, and optionally solidifying agent,
In yet another embodiment of the present invention, fermenting the inoculated culture at temperature about 27-30xc2x0 C., and
In still another embodiment of the present invention, obtaining large quantity of viable spores.
In still another embodiment of the present invention, inoculating said culture of step (a) with said spores of final concentration of about 1xc3x97106 conidia/ml.
In still another embodiment of the present invention, fermentation said culture of step (b) under solid state, surface, or submerged fermentation conditions.
In still another embodiment of the present invention, wherein rotating said inoculated culture in a rotor at rate of about 200 to 400 revolutions/minute.
In still another embodiment of the present invention, wherein agitating the said culture with said rotor at about 1.5-3.5 c.m. throw.
In still another embodiment of the present invention, wherein fermenting said culture with aeration rate of about 0.5-1.0 vvm.
In still another embodiment of the present invention, wherein fermenting said culture with pressure of about 07-1.4 psig.
In still another embodiment of the present invention, wherein spores of step (c) show viability ranging between 90 to 100%.
In still another embodiment of the present invention, wherein mixing said culture with a carrier in a ratio ranging between 1:2 to 2:1, to develop commercial formulation with biocontrol activity.
In still another embodiment of the present invention, wherein carrier used is lignite.
In still another embodiment of the present invention, said method show most efficient sporulation with composition comprising molasses about 7.50 g/L, Corn Steep Liquor (CSL) about 20 g/L, Sodium chloride (NaCl) about 10 g/L, calcium sulphate (CaSO4) about 0.25 g/L, Potassium dihydrogen phosphate (KH2PO4) about 0.0060 g/L, Magnesium sulphate (MgSO4.7 H2O) about 0.0050 g/L, Copper sulphate (CuSO4) about 0.0010 g/L, Ferrous sulphate (FeSO4) about 0.0016 g/L, an anti-foam agent, and optionally solidifying agent.
In still another embodiment of the present invention, said method shows duration of viability of spores of about 10-14 months at temperature of about 4xc2x0 C. with carriers.
In still another embodiment of the present invention, said method shows duration of viability of spores of about 4-8 months at temperature of about 30xc2x0 C. with carriers.
In still another embodiment of the present invention, said method shows average spore count ranging between 1-5xc3x97108 conidiospores/ml.
In still another embodiment of the present invention, development of a novel medium based on inexpensive agro-based industrial by-products like beet-root pulp, wheat bran, corn-steep liquor, molasses and some inorganic salts in a definite proportion useful for the production of viable spores of Trichoderma species (used as biocontrol agents) and a number of other sporulating ascomycetous fungi such as Aspergillus species, Penicillium species and other scantily sporulating species of fungi under solid state, surface and submerged fermentation conditions.
In still another embodiment of the present invention, conventional processes take 5-9 days for the production of 10 fold increase in the number of spores of Trichoderma species from the initial inoculum as against in the present novel medium wherein 100 fold increase in the number of spores is achieved in 48 hours.
In still another embodiment of the present invention, this therefore makes the present invented process an economical, fast and highly advantageous from the commercial viewpoint.
In still another embodiment of the present invention, ingredients based on agro-waste industrial by-products are high energy and protein rich natural sources containing large number of trace elements and are very often used in culture media. However, these ingredients have not been used for inducing early sporulation in ascomycetous fungi. These ingredients in the medium result in very fast and better sporulation under submerged fermentation conditions when compared to conventional medium (Molasses-yeast extract medium) containing yeast extract, peptone, malt extract and refined sugars like glucose, and sucrose.
In still another embodiment of the present invention, the above ingredients were dissolved in de-ionised water and steam sterilised at 121xc2x0 C./15 lb for 15 min. The sterilisation of the medium is done ex-situ for the operation of small bench fermentors and in-situ when large vessels are operated.
In still another embodiment of the present invention, for initiation of the fermentation process, spores from the a week old culture of Trichoderma species raised on solid medium (FM1medium with agar) are eluted with pre-sterile Tween 80 solution (0.02% v/v in water) and inoculated in the fermentor to the final concentration of 1xc3x97106 conidia/ml.
In still another embodiment of the present invention, fermentation is carried out in FM1 medium in cylindrical glass jars or stainless steel vessels at 29xc2x11xc2x0 C.
In still another embodiment of the present invention, the vessels are agitated with standard stirrers at a moderate rate of 200-400 rpm and aerated at a constant air flow of 0.5 to 1.0 volumes per volume of liquid per minute at 1 bar (kg) over pressure.
In still another embodiment of the present invention, all the inoculated spores germinate and vegetative growth of the fungus with a numerous chlamydospores emerges within 24-h fermentation period.
In still another embodiment of the present invention, thereafter, sporulation sets in and chiamydospores disappear completely within next 24-h fermentation period.
In still another embodiment of the present invention, fermentation is terminated after an incubation period of 48-72 h or until the maximum conidiospore concentration of 1-5xc3x97108/ml is obtained and no chlamydospores are spotted under microscopic examination.
In still another embodiment of the present invention, whole culture broth is thereafter thoroughly mixed with pre-sterilised lignite (dry heated at 120xc2x0 C. for 6-8 h. and later cooled to room temperature) of mesh size 200-250 in the ratio of 1:2 and packed in 200 g packets in milky white pollywogs (size 6xe2x80x3xc3x9710xe2x80x3) of 50-75 xcexcg thickness.
In still another embodiment of the present invention, the biocontrol formulation thus prepared can be stored at 4xc2x0 C. for a period of 12 months and at 30xc2x0 C. upto 6 months, without significant loss of viability.
It is surprising to notice sporulation of this exorbitant quantum in fungi and that too in such a short span of time. The said composition shows about 100-fold increase in spore count within 48 hours of inoculation. All the ingredients of the composition comprising molasses ranging between 5-20 g/L, Corn Steep Liquor (CSL) ranging between 10-25 g/L, Sodium chloride (NaCl) ranging between 5-15 g/L, calcium sulphate (CaSO4) ranging between 0.1-0.5 g/L, Potassium dihydrogen phosphate (KH2PO4) ranging between 0.001-0.01 g/L, Magnesium sulphate (MgSO4.7H2O) ranging between 0.001-0.01 g/L, Copper sulphate (CuSO4) ranging between 0.001-0.0050 g/L, Ferrous sulphate (FeSO4) ranging between 0.0009-0.005 g/L, an anti-foam agent, and optionally solidifying agent, show synergy to bring about unexpected early and profuse sporulation.
The said ingredients and their quantum in said composition are critical. Any change in the ingredients or their quantity do not produce desired results. In one experiment, molasses was not added in the said composition and sporulation was observed with only about 5-fold increase. Similarly, not adding corn-steep liquor led to only about 4-fold increase in the sporulation. Also, when all the said ingredients of the said composition are added but with potassium dihydrogen phosphate at concentration higher then the above-mentioned range, sporulation was only about 2-fold. Similarly, higher then the above defined range of copper sulphate showing normal (normal means using conventional substrate) sporulation with no increase in sporulation at all.
All these observations prove that all the said ingredients and their concentration ranges are extremely critical for the desired results. Any deviation from the same severely affects the sporulation in fungi.
The Following examples are given by way of illustration and should not construe the scope of the invention.